Insulin-stimulated glycogen synthase (GS) activity of in human muscle correlates with insulin-mediated glucose disposal rates and is reduced in insulin-resistant subjects. Activation of protein phosphatase-1 (PP-1) may contribute to the mechanism by which insulin activates GS. We investigated the change of phosphorylase phosphatase (PHP) and GS activities during a 2-hour hyperinsulinemic euglycemic clamp in 12 insulin-sensitive (group S) and 8 insulin-resistant (group R) subjects. Muscle biopsies were obtained from quadriceps femoris muscle at times 0, 10, 20, 40, and 120 minutes of insulin infusion. GS fractional activity was increased significantly by 10 minutes in group S, and by 20 minutes in group R but remained low compared to group S at 120 minutes. Fasting PHP activity was low for group R compared to group S, and did not increase significantly in group R until 20 minutes. In group S, PHP was significantly stimulated by 10 minutes, remaining significantly higher than in group R at all time points. The insulin-mediated changes in phosphatase activities were not decreased by 3 nM okadaic acid (OA) but were completely inhibited by 1 uM OA verifying insulin activation of a type 1 phosphatase. Subcellular fractionation demonstrated reduced fasting PP-1 activities in both the glycogen and cytosolic fractions of muscle obtained from subjects in group R compared to those in groups S. These results suggest that activation of PP-1 could contribute to insulin-- stimulation of GS in human muscle. Lower fasting PP-1 in cytosol and glycogen subcellular fraction plus low insulin stimulated PP-1 activity could explain, in part, reduced insulin stimulated GS in insulin-resistant subjects.